NOX2-TRPM2 coupling promotes Zn2+ inhibition of complex III to exacerbate ROS production in a cellular model of Parkinson’s disease

Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by the limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinson’s disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP+ (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (1) MPP+ stimulates ROS production by raising cytoplasmic Ca2+ levels, rather than acting directly on mitochondria. (2) To raise the Ca2+, MPP+ co-stimulates NADPH oxidase-2 (NOX2) and the Transient Receptor Potential Melastatin2 (TRPM2) channel that form a positive feedback loop to support each other’s function. (3) Ca2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn2+. (4) Zn2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca2+ and Zn2+, and prevention of electron escape from complexes -all abolished the ability of MPP+ to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP+, while Zn2+ ionophore replicated the effects of both MPP+ and Ca2+. Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca2+, Zn2+, and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases.

www.nature.com/scientificreports/However, antioxidant supplements have yielded disappointing outcome in clinical trials 2,6,8 .This lack of success could be attributed to the inability of antioxidants to discriminate pathogenic ROS from physiological ROS [6][7][8][9] .Furthermore, scavenging of pathogenic .OH is thought to be impractical 8 .Thus, to develop safe and effective redox medicines, it is imperative to gain a better understanding of how the shift from physiological to pathological ROS status occurs.
However, ROS biology is far too complex, involving multiple signals and mechanisms.One key signal is Ca 2+10,11 .Indeed, there is a direct correlation between the cytosolic Ca 2+ rise and the amount of ROS produced 10,11 .In healthy cells, physiological stimuli induce a modest rise in cytosolic Ca 2+ to stimulate ROS production at levels appropriate for redox signalling.Pathogenic insults, however, rise the Ca 2+ to supraphysiological concentrations to bolster ROS production to toxic levels 10,11 .ROS are produced from multiple sources, including mitochondrial complexes and dehydrogenases, and extra-mitochondrial enzymes, such as NADPH oxidases (NOXs) 1,2,6,8,10,12 .Furthermore, ROS produced at one site can impact ROS generation at a different site-a phenomenon known as ROS-induced ROS production 12,13 , which adds an additional layer of complexity to ROS homeostasis.
Regulation of Ca 2+ homeostasis is equally complex, involving various ion channels, exchangers and transporters that transport Ca 2+ between the Ca 2+ rich compartments and cytoplasm 10,11 .The picture gets even more complex by the existence of a Ca 2+ -ROS feedback cycle, where ROS can trigger cytosolic Ca 2+ rise and Ca 2+ , in turn, can stimulate ROS production [10][11][12] .The precise mechanisms underlying the Ca 2+ -ROS cycle are not fully understood but appear to involve an intricate interplay between ROS-sensitive calcium channels and Ca 2+ -dependent ROS-generation from NADPH oxidases (NOXs) and mitochondria 10 .Some Ca 2+ channels are ROS sensitive and therefore can contribute to Ca 2+ -ROS interplay 10 .Among them, Transient Receptor Potential Melastatin2 (TRPM2) calcium channels are important.These channels are coactivated by ROS-generated ADPR (ADP-ribose) and Ca 2+14,15 .Importantly, activation of these channels has been linked to numerous diseases where ROS are upregulated 16,17 .Therefore, we hypothesise that TRPM2 channels play a central role in Ca 2+ -ROS interplay.
To test this hypothesis, we utilized a well-established cellular model of Parkinson's disease (PD) 18 as an exemplar of ROS-linked diseases.We exposed SH-SY5Y neuroblastoma cells to the PD-causing neurotoxin MPP + (1-methyl-4-phenylpyridinium) to upregulate Ca 2+19 and investigated how the rise in Ca 2+ augments ROS production leading to cell death.Our data reveal a distinct signalling circuit -comprising NOX2, the TRPM2 channel, Ca 2+ , Zn 2+ , and respiratory complexes I and III, predominantly the latter-that links Ca 2+ to pathogenic levels of ROS production.

Amplification of mitochondrial ROS is essential for the neurotoxic effect of MPP +
We first investigated the relative contributions of mitochondrial and extra-mitochondrial sources to MPP + -induced cell death.For this, we exposed SH-SY5Y cells to 1 mM MPP + for 24 h and assessed ROS production and cell viability (for optimisation of conditions, see Supplemental Fig. 1).We used dihydroethidium (DHE) to detect total ROS from all cellular sources, including mitochondria, and MitoSOX Red to specifically detect mtROS.MPP + treatment significantly increased the fluorescence of both the reporters.The total ROS signal was abolished by the general ROS quenchers, N-acetylcysteine (NAC) and TEMPO (Fig. 1a,b), confirming that the increase in fluorescence is due to ROS.Interestingly, Mito-TEMPO, a ROS quencher specific for mtROS, was able to abolish the total ROS signal almost as effectively as the general ROS quenchers (Fig. 1a-c).Furthermore, Mito-TEMPO was able to abolish MPP + -induced cell death (Fig. 1d,e).These results are seemingly in agreement with the numerous reports that MPP + acts directly on mitochondria to produce cytotoxic ROS 20 .

MPP + upregulates mitochondrial ROS production indirectly by generating Ca 2+ and Zn 2+ signals
It is widely believed that MPP + acts directly on respiratory complex I to generate mtROS 20,21 .However, MPP + has also been shown to elevate cytoplasmic Ca 2+19 .Since Ca 2+ is a known stimulant of mtROS 10,11 , we asked whether MPP + -induced mtROS production could, in part, be Ca 2+ mediated.As expected, MPP + treatment caused an increase in intracellular Ca 2+ levels (Fig. 2a,b).Rather unexpectedly, chelation of Ca 2+ with BAPTA (1,2-bis-(aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester) completely abolished the ability of MPP + to stimulate mtROS production (Fig. 2c-e).These data suggested that the MPP + -induced mtROS production is almost entirely mediated by Ca 2+ , and/or other metal ions that BAPTA is capable of binding to.We considered Zn 2+ as the other metal ion because its affinity for BAPTA is greater than that for Ca 2+ , and Zn 2+ is a known stimulant mtROS production 22 .Thus, we tested the effect of the Zn 2+ chelator, TPEN (N,N,N′,N′tetrakis (2-pyridylmethyl) ethylenediamine), using a low concentration to chelate Zn 2+ , while excluding Ca 2+23 .Remarkably, TPEN was as effective as BAPTA in suppressing MPP + -induced ROS production (Fig. 2c-e) and the consequent cell death (Fig. 2f,g).The ability of the two chelators to completely abolish MPP + -induced ROS production argues against the numerous reports that MPP + acts directly and solely on mitochondria 24,25 .Instead, our results suggest that MPP + depends on Ca 2+ and Zn 2+ to stimulate ROS from mitochondria.

TRPM2-mediated Ca 2+ rise upregulates mitochondrial ROS to cytotoxic levels
We next set out to investigate the mechanism by which MPP + raises cytoplasmic Ca 2+ levels.A previous study has shown that MPP + can stimulate Ca 2+ entry via TRPM2 channels 19 .However, neuronal cells have multiple mechanisms to regulate Ca 2+26 .Thus, it was not clear whether TRPM2-mediated Ca 2+ entry alone can account for MPP + induced ROS production.Accordingly, we tested the effect of inhibiting TRPM2-mediated Ca 2+ influx on ROS production.Inhibition of TRPM2 channels with three pharmacological inhibitors (PJ34, 2-APB, and ACA) with different modes of action 27 abolished MPP + -induced mtROS production (Fig. 3a-c) and cell death (Fig. 3d-e).2-APB and ACA are thought to inhibit the channel directly, whereas PJ34 inhibits the production of the activator of TRPM2 channel by inhibiting poly-ADP-ribose polymerase.Likewise, silencing RNA targeted to the TRPM2 channel, but not the control scrambled siRNA, abrogated ROS production (Fig. 3f-h) and cell death (Fig. 3i,j).Importantly, there was no significant difference between control cells and cells exposed to MPP + but had been pre-treated with TRPM2 inhibitors/siRNA (Fig. 3b,c,g,h).These results imply that TRPM2 mediated Ca 2+ entry fully accounts for the MPP + -induced ROS production and has little or no effect on basal ROS production.
To seek independent evidence for the exclusive role of TRPM2 in mtROS generation, we used HEK293 cells stably transformed with the FLAG-tagged human TRPM2 cDNA placed under the control of a tetracyclineregulated promoter (HEK293-TRPM2 tet ) 28,29 .Western blotting confirmed the expression of TRPM2 channels in tetracycline induced, but not in non-induced, cells (Fig. 4a; Supplementary Fig. 2).Functional analysis showed that H 2 O 2 was able to induce robust 2-APB sensitive Ca 2+ influx in the induced, but not non-induced, cells (Fig. 4b).Using these validated cells, we demonstrated that H 2 O 2 had virtually no effect on ROS production in uninduced cells but triggered robust ROS production in induced cells that was sensitive to 2-APB inhibition (Fig. 4c-e).
Finally, we used a non-neuronal cell line, the pancreatic β-cell line (INS-1 832/13) that natively expresses TRPM2 channels.These cells are sensitive to palmitate induced nutrient stress and have been used as a cellular model for obesity-induced type-2 diabetes 29 .The results show rescue of palmitate-induced mtROS production by both the pharmacological inhibitor PJ34 (Fig. 4f,g) and TRPM2 siRNA (Fig. 4h,i).In addition, TPEN attenuated palmitate-induced mtROS production (Fig. 4f,g), indicating that the role of Zn 2+ is not limited to neuronal cells.
Thus, using three different cellular models and three different stressors, we demonstrated that TRPM2mediated Ca 2+ entry upregulates mtROS production to cytotoxic levels, but has little or no impact on basal ROS production.Thus, TRPM2 dependent pathogenic ROS production appears to be not unique to neuronal cells but shared by other cell types.Co-incidentally, the absolute dependence of MPP + on TRPM2 channels for mtROS overproduction further excludes the direct action of MPP + on mitochondria.

TRPM2-mediated Ca 2+ influx requires simultaneous activation of NADPH oxidase-2
TRPM2 channels require ROS for activation.Given the above evidence that MPP + cannot directly stimulate ROS from mitochondria (Figs. 2, 3 and 4), we asked whether MPP + could target NOX2 30 to generate the ROS required for TRPM2 activation.Consistent with this idea, inhibition of NOX2 with apocynin (a pan NOX inhibitor) and gp91-ds-tat (a NOX-2-specific inhibitor) prevented MPP + -induced Ca 2+ rise (Fig. 5a,b), and the consequent ROS production (Fig. 5c-e) and cell death (Fig. 5f,g).These findings support the idea that NOX2 activation likely provides the ROS necessary for TRPM2 activation and the subsequent mtROS upregulation and cell death.
Taken together, these results suggest that simultaneous activation of NOX2 and TRPM2 channels is necessary for MPP + to generate the Ca 2+ signals required for mtROS production and cell death.

Zn 2+ acts downstream of Ca 2+ to augment mitochondrial ROS production to cytotoxic levels
While the above data (Figs.2, 3 and 4) are entirely consistent with the established role that Ca 2+ plays a role in mtROS production, they fail to explain why the Zn 2+ chelator was as effective as the Ca 2+ chelator in suppressing mtROS production (Fig. 2).To address this, we postulated a hierarchical relationship between the two cations, with Zn 2+ acting downstream of Ca 2+ .
To test the hypothesis, we raised the intracellular Ca 2+ by treating the cells with the calcium ionophore A23187.We have included the membrane impermeable Zn 2+ chelator DTPA (diethylenetriaminepentaacetic acid) during the treatment to exclude unwarranted Zn 2+ entry.A23187 promoted cytosolic Ca 2+ rise (Fig. 6a,b), increasing the ROS production (Fig. 6c-e) and cell death (Fig. 6f,g).However, TPEN was able to mitigate all    www.nature.com/scientificreports/these effects as effectively as BAPTA (Fig. 6c-g).These data support the hypothesis that Zn 2+ acts downstream of Ca 2+ to upregulate cytotoxic mtROS production.
To confirm that Zn 2+ is the ultimate ROS-inducing signal, we elevated the intracellular Zn 2+ levels with the zinc ionophore, zinc-pyrithione (ZnPTO).Ca 2+ was excluded from the treatment medium to avoid any confounding effects from Ca 2+ entry.ZnPTO treatment caused a rise in intracellular free Zn 2+ (Fig. 6h,i) and has increased mtROS production (Fig. 6j,k) and cell death (Fig. 6l,m), the effects being rescued by TPEN (Fig. 6j-m).Thus, raising the intracellular Zn 2+ was able to faithfully phenocopy the effects of Ca 2+ elevation induced by MPP + and A23187.
Taken together, our results demonstrate that during oxidative stress, rise in cytosolic Ca 2+ alone is not enough to augment cytotoxic mtROS production, but deployment of Zn 2+ as a downstream signal is essential.

Zn 2+ inhibition of complexes I and III accounts for MPP + (Ca 2+ )-induced mitochondrial ROS upregulation
We next asked how the MPP + -generated ionic signals exacerbate mtROS production.Although mitochondria have more than ten ROS generating sites, complexes I and III produce the majority (~ 90%) of mtROS 1-3 .We therefore examined the roles of these two complexes using compounds that selectively suppress electron leak from complex I (S1QEL) 31 and complex III (S3QEL) 32 .Both compounds attenuated MPP + -induced mtROS production (Fig. 7a,b) as well as cell death (Fig. 7c,d).However, S3QEL was more effective than S1QEL, even though its reported EC 50 value in the ROS assay (~ 1.75 µM) is more than one order of magnitude greater than that of S1QEL (~ 0.06 µM) 31,32 .
We next substituted ionophores for MPP + to determine the effects of Ca 2+ and Zn 2+ on the complexes.As with MPP + , both the QEL compounds suppressed the effects of A23187 on mtROS production (Fig. 7e,f) and cell death (Fig. 7g,h); these effects of QEL compounds are very much akin to TPEN (Fig. 7e-h).Likewise, the QEL compounds were able to mitigate ZnPTO-induced mtROS production (Fig. 7i,j) and cell death (Fig. 7k,l).However, while S3QEL appeared to be slightly more effective than S1QEL, this difference did not reach statistical significance, presumably because ionophores raise the metal ion concentrations to levels well beyond what the pathogenic insults can achieve.
Collectively, these data suggest that MPP + exacerbates mtROS production by generating Ca 2+ -driven Zn 2+ signals that act on complexes I and III, primarily complex III (Fig. 7m).Importantly, our data provide a fundamental new insight into how Ca 2+ affects ROS production: while Ca 2+ can increase ROS production due to its ability to increase in electron transport through the complexes 10,11 , for it to escalate ROS production to cytotoxic levels Zn 2+ participation is mandatory.

Discussion
Here we report the discovery of a signalling circuit that deciphers the complex mechanism underlying the production of pathogenic levels of mtROS.Although several signalling molecules including Ca 2+10,11,33 , mitochondrial complexes 1,6,7,34-36 and NOX2 13,25,37 have been implicated, the question of whether and how they interact to drive mtROS production remained unclear.To address this, we have used a cellular model of PD 18 wherein we exposed the SH-SY5Y cells to the PD causing MPP + toxin and monitored the signalling events that upregulate mtROS production.The findings revealed that Ca 2+ , NOX2 and respiratory complexes form a signalling circuit by engaging TRPM2 channels and Zn 2+ ions.Our study reveals several fundamental insights into the mechanism.First, MPP + co-activates TRPM2 channels and NOX2 to rise cytoplasmic Ca 2+ .Second, Ca 2+ targets mitochondrial complexes I and III to produce almost all pathogenic ROS; however, contrary to the widely held view 10,11,33 , the Ca 2+ effect is not direct, but is mediated by Zn 2+ .Third, the signalling circuit plays no role in basal ROS production but is activated during external stress to drive ROS production to cytotoxic levels (depicted schematically in Fig. 8).Although MPP + can increase cytoplasmic Ca 2+ in SH-SY5Y cells 19 (Fig. 2a), Ca 2+ has not been implicated in MPP + induced ROS production.Instead, the current notion is that the toxin inhibits complex I directly to stimulate ROS production 38 .Although this view is based on studies with isolated mitochondria 24,38 and has been challenged by in vivo studies where knock-out of complex I failed to prevent dopaminergic neuronal loss 39 , the historic view continues to prevail due to lack of mechanistic evidence.Given that Ca 2+ is known to signal mtROS production 10,11,33 , we asked whether the ROS-inducing effect of MPP + is, at least in part, owing to its ability to rise cytoplasmic Ca 2+ .Indeed, chelation of Ca 2+ with BAPTA abolished the ability of MPP + to augment cytotoxic mtROS production (Fig. 2), suggesting that the MPP + effect is almost entirely mediated by Ca 2+ .However, BAPTA is not specific for Ca 2+ , but binds other metal ions, especially Zn 2+ which has been shown to upregulate mtROS production in neuronal cells 22 .Indeed, chelation of Zn 2+ with TPEN, using a concentration (1 µM) too low to chelate Ca 2+ , was as effective as BAPTA in mitigating MPP + induced ROS production (Fig. 2).Thus, we conclude that the ability of MPP + to trigger cytotoxic mtROS is not due to its direct effect on complex I, rather it is mediated by Ca 2+ and Zn 2+ ions.

MPP + induced Ca 2+ influx requires simultaneous activation of TRPM2 channels and NADPH oxidase-2
We next asked how MPP + increases cytoplasmic Ca 2+ .Although neuronal cells have multiple mechanisms to regulate Ca 2+11 , inhibition of Ca 2+ influx through TRPM2 channels alone was sufficient to abolish MPP + -induced mtROS overproduction (Fig. 3), indicating the unique role TRPM2 channels play in mtROS production.However, TRPM2 channels, paradoxically, require ROS for activation 27,40 .We demonstrate that ROS required for TRPM2 activation comes from NOX2 30 activation (Fig. 5).The finding that NOX2 inhibitors (Fig. 5) are as effective as TRPM2 inhibitors (Figs. 3 and 4) in mitigating MPP + -induced Ca 2+ influx, and the subsequent mtROS production, raise the possibility that synchronised activation of NOX2 and TRPM2 likely occurs during MPP + intoxication.Such mutual cooperation would allow NOX2 to provide the ROS necessary for TRPM2 activation, and TRPM2 activation to supply Ca 2+ for NOX2 activation 37 .Such functional interplay between TRPM2 and NOX2 potentially establishes a positive feedback loop to amplify the Ca 2+ signals required for mtROS upregulation.
Although molecular basis for the interplay remains to be determined, it is interesting to note that upregulation of both NOX2 13,37 and TRPM2 channels 16,17 has been reported in numerous ROS-associated linked diseases.Furthermore, pharmacological, or genetic intervention of NOX2 13,37 and TRPM2 channels 16,17 produces similar positive outcomes in experimental models of numerous ROS-linked diseases.Notably, activity of PARP1-an enzyme that produces ADPR -an endogenous activator of TRPM2 channels 15,27,40 -is markedly upregulated in several neurodegenerative diseases, including PD and Alzheimer's disease 41,42 .It therefore seems likely that co-activation of NOX2 and TRPM2 channels is a common mechanism shared by most ROS-linked diseases.
We next asked how Zn 2+ plays as an important role as Ca 2+ in upregulating mtROS.To address this, we hypothesised a hierarchical relationship between the two signalling ions, with Zn 2+ acting downstream of Ca 2+ .Consistent with this hypothesis, calcium ionophore-induced cytosolic Ca 2+ rise induced mtROS production, yet this effect was entirely abrogated by TPEN (Fig. 6).Moreover, elevation of intracellular Zn 2+ with the ionophore, ZnPTO, led to mtROS production independently of Ca 2+ (Fig. 6).Collectively, these data argue that when cytoplasmic Ca 2+ levels reach supraphysiological concentrations, Zn 2+ acts downstream of Ca 2+ to drive mtROS production to pathogenic levels.
Numerous reports have linked Zn 2+ to PD pathology.Frist, Zn 2+ is recognised as an environmental risk factor for PD 43 .Second, accumulation of free Zn 2+ is a key feature of degenerating dopaminergic neurons 44 with a recent machine-learning study placing Zn 2+ accumulation as a top predictor of PD 45 .Third, post-mortem examination of brains of PD patients 46 , Lewy body-injected macaque monkeys 45 and MPP + intoxicated mice 44 display deposits of labile Zn 2+ .Fourth, zinc transporters are upregulated in the brains of PD patients 45 .Fifth, Zn 2+ chelators protect mice against neurodegeneration in substantia nigra in in vivo models 47 .Finally, mutations in the PD associated PARK9 gene disrupt Zn 2+ as well as ROS homeostasis 48 .Thus, our findings provide a mechanistic explanation for the well-documented, yet poorly understood role of Zn 2+ in PD pathogenesis.
It is likely that in other ROS-driven diseases, where Ca 2+ is implicated, Zn 2+ plays a downstream role in generating pathogenic ROS.The beneficial effects of Zn 2+ chelators in some experimental disease models including neurodegenerative 47,49 , 50 and metabolic diseases 51 lend some support to this plausibility.

Zn 2+ inhibition of complexes I and III accounts for MPP + (Ca 2+ )-induced mitochondrial ROS upregulation
Guided by the previous reports that Zn 2+ can inhibit complexes I and III 52,53 , we asked whether MPP + , via Zn 2+ , targets these complexes.We first demonstrated that MPP + causes a rise in mitochondrial Zn 2+ (Supplemental Fig. 3), that is dependent on TRPM2 activation (Supplemental Figs. 3 and 4).Using compounds that selectively quench electron escape from complex I (S1QEL) and complex III (S3QEL) 7,32 , we demonstrate that MPP + targets both complex I and III to induce mtROS production (Fig. 7).These compounds suppressed MPP + -induced mtROS production, as well as mtROS induced by the calcium and zinc ionophores.These findings support the idea that the ability of MPP + to induce excess mtROS stems from its ability to generate the Zn 2+ signals that act on mitochondrial complexes.Our data showed that the MPP + effect is more pronounced on complex III (Fig. 7).This is presumably due to the much greater affinity of Zn 2+ for complex III (Ki = 0.1 µM) 52,53 compared with complex I (IC 50 10-50 µM) 53 .This is consistent with the presence of a validated Zn 2+ inhibitory binding residue, E within the catalytic centre of complex III, containing the highly conserved PEWY motif 54 .Complex I appears to lack Zn 2+ sensitive residues within the catalytic centre, but Zn 2+ was found in the regulatory 13 KDa accessory subunit of the complex 55 , however with no demonstrated role.
We consider the above findings in the context of pathophysiology.Under physiological conditions, the concentration of mitochondrial Zn 2+ is too low (~ 60 pM) 56 to stimulate ROS from complexes I and III.However, basal ROS production continues to occur due to the ability of Ca 2+ to stimulate electron flow and thereby increase ROS production from the complexes as well as other sites in mitochondria.During chronic stress, mitochondrial Zn 2+ uptake increases, leading to the inhibition mostly of complex III due to its high affinity for Zn 2+ .From a pathological perspective, targeting complex III would seem advantageous because complex III releases O 2 .-intowww.nature.com/scientificreports/ the intermembrane space, from where it can more readily escape into the cytoplasm compared to the ROS released into the mitochondrial matrix from complex I 57 .Consistent with the dominant role of complex III, genetic depletion of the Ndufs4 gene, which encodes a functional subunit of complex I, failed to prevent MPP + -induced ROS generation and death of dopamine-secreting neurons 39 .Furthermore, a recent study reported a link between autosomal dominant mutations in UQCRC1, encoding a subunit of complex III, and familial Parkinson's disease 58 .These findings collectively suggest that the role of complex I in generating pathogenic levels of mtROS in PD, relative to complex III, may have been exaggerated in the literature.

Limitations of the study and future directions
Although these data were obtained with the widely used cellular model of PD, and several of the findings would appear to be in general agreement with the clinical features and in vivo studies of PD, these findings need to be validated using appropriate genetic and pharmacological animal models and human organoid cultures.Such studies could explore the use of genetically encoded fluorescent reporters of metal ions and ROS.From a mechanistic perspective, it is important to elucidate (1) how NOX2 and TRPM2 channels communicate with each other, (2) how the resultant rise in cytosolic Ca 2+ affects Zn 2+ homeostasis, and (3) how Zn 2+ enters mitochondria during oxidative stress.Furthermore, data from pancreatic β-cells and the recombinant HEK cells presented in the present study would suggest that the mechanism revealed is likely shared by numerous other diseases.Thus, future studies could be directed to investigate this possibility.

Conclusions and implications of the study
In summary, our findings present novel mechanistic insights into the upregulation of mtROS in a well-established cellular model of PD.Our work unravelled a signalling circuit encompassing NOX2, TRPM2, Ca 2+ , Zn 2+ and complexes I/III.The presence of excessive NOX2 13 , Zn 2+59 , and oxidatively damaged biomolecules 60 in the postmortem brains of PD patients, coupled with the genetic association between complex III and familial PD 58 , underscore the pathological relevance of this signalling circuit.Interestingly, toxic aggregates of α-synuclein have been shown to induce Ca 2+ influx 61 , raising the possibility that the pathological effects of α-synuclein could be due to activation of the same signalling circuit.Although our findings were made using the cellular models of PD and diabetes, it is likely that this signalling circuit operates in a multitude of other conditions where mitochondrial dysfunction is a common denominator; these include other neurodegenerative diseases, metabolic diseases including diabetes 5,6 , and perhaps ageing 62 .The fact that the signalling circuit is selectively activated by pathogenic insults suggests that it may represent a safe and effective therapeutic target for PD and a host of other diseases sharing common mechanisms.

Treatments
Cells were plated on poly-L-lysine (A003, Millipore; 50 µg/ml) coated 96-well plates and grown in complete medium for 24 h to ~ 50% confluency.Cells were treated with the desired reagent prepared in the complete medium from stock solutions for the desired length of time at 37 °C (see figure legends for specific details).Stock solutions were prepared as follows: 1-Methyl-4-phenylpyridinium (MPP + ) iodide (D048, Sigma-Aldrich),

Cell viability assays
Following the treatment with the desired reagents, cells were washed with Hanks Balanced Salt solution (HBSS, 17420014, Corning) and stained with HBSS containing propidium iodide (5 µg/ml; P4170, Sigma-Aldrich) and Hoechst 33342 (4 µM, 5117, TORCIS Bioscience) for 30 min.Stained cells were imaged using an epifluorescent microscope (EVOS FL Auto Imaging System; Thermo Scientific Invitrogen).Percent cell death was calculated from the ratio of propidium iodide (PI) to Hoechst-stained cells.

ROS detection
Total ROS was detected using H
After taking basal readings, 20 µl of 15 mM H 2 O 2 (22460250, Acros Organics) was injected into each well and the recordings continued.
To detect Ca 2+ changes in SH-SY5Y cells, cells were grown in µ-Slide 8 well high ibiTreat (80,806 Thistle Scientific) or 96-well plates to approximately 60% confluency and preloaded with 1 µM Fluo4-AM (F14201, Invitrogen)/0.02%Pluronic F127 in HBSS.Following treatment with vehicle or test reagents at 37 °C for the desired length of time, fluorescence was captured using the LSM700 inverted confocal microscope (excitation 494 nm; emission, 519 nm) or EVOS FL Auto 2 Imaging System (using a GFP filter).

Data analysis and presentation
Fluorescence intensity (arbitrary units, A.U) of cells stained with various fluophores (DCF, DHE, MitoSOX, Fluo-4 and FluoZin-3) was estimated using Image J as described before 28 .All experiments were performed at least three times (n, biological replicates), each in duplicate (technical replicates) wells.Images were acquired from three random fields of each well.Fluorescence intensity values were normalised to the number of cells determined by counting the Hoechst-counter stained nuclei.Data are presented in bar charts as mean ± S.E.M. Statistical significance was determined using the One-way ANOVA (Origin), followed by Tukey's posthoc test; probability (P) values are indicated with *, **, *** and **** which correspond to values of 0.05, 0.01, 0.001, and 0.0001 respectively.

Figure 1 .
Figure 1.MPP + exacerbates mitochondrial ROS production to cause SH-SY5Y cell death.(a-c) MPP + induces ROS production.(a) Fluorescent images of SH-SY5Y cells stained for total (DHE) and mitochondrial (MitoSOX) ROS, recorded after 24-h treatment with medium alone or medium containing 1 mM MPP + ± the indicated antioxidants (5 mM NAC, 10 µM TEMPO or 10 µM Mito-TEMPO); scale bar = 200 µm.(b and c) Mean ± SEM of fluorescence intensity corresponding to (a).(d and e) MPP + induces cell death.(d) Fluorescent images of cells treated as in (a) but stained for cell death (Hoechst and PI); scale bar = 125 µm.(e) Mean ± SEM of % PI positive dead cells corresponding to (d).Mean data in (b, c and e) are from three independent experiments (n = 3); * p ˂ 0.05, ** p ˂ 0.01; *** p ˂ 0.001 from one-way Anova, post-hoc Tukey test.(f) Schematic summary of findings.

Figure 2 .
Figure 2. MPP + upregulates intracellular Ca 2+ and Zn 2+ but chelation of Zn 2+ alone is sufficient to prevent mtROS overproduction and cell death.(a and b) MPP + causes a rise in intracellular Ca 2+ .(a) Fluorescent images of SH-SY5Y cells stained for Ca 2+ with Fluo-4 imaged after 24-h treatment with medium alone or medium containing 1 mM MPP + ; scale bar = 75 µm.(b) Mean ± SEM of fluorescence intensity corresponding to (a).(c-e) Chelation of Zn 2+ is as effective as Ca 2+ chelation in attenuating ROS production.(c) Fluorescent images of cells stained for total (DHE) and mitochondrial (MitoSOX) ROS, imaged after 24-h treatment with medium alone or medium containing 1 mM MPP + ± BAPTA-AM (5 µM) or TPEN (0.5 µM); scale bar for DHE = 125 µm; MitoSOX = 75 µm.(d and e) Mean ± SEM of fluorescence intensity corresponding to (c).(f and g) Chelation of Zn 2+ is as effective as Ca 2+ chelation in preventing MPP + -induced cell death.(f) Fluorescent images of cells treated as in (c) but stained for cell death (Hoechst and PI); scale bar = 125 µm.(g) Corresponding mean ± SEM of % PI positive dead cells.Mean data in (b, d, e and g) are from three independent experiments (n = 3); **p ˂ 0.01; ***p ˂ 0.001 from One-way Anova, post-hoc Tukey test.(h) Schematic summary of findings.

Figure 5 .
Figure 5. NOX2 inhibition prevents MPP + -induced intracellular Ca 2+ rise, mitochondrial ROS production and cell death.(a and b) NOX2 inhibition prevents MPP + -induced Ca 2+ rise.SH-SY5Y cells were treated with medium alone, or medium containing 1 mM MPP + ± NOX inhibitors (10 µM apocynin and 5 µM gp91ds-tat) for 24 h.(a) Fluorescent images of cells stained for Ca 2+ with Fluo-4; scale bar = 125 µm.(b) Mean ± SEM of fluorescence intensity corresponding to (a).(c-e) NOX2 inhibition prevents MPP + -induced increase in total as well as mitochondrial ROS production.Cells were treated as in (a) and stained for ROS.(c) Fluorescent images of cells stained for total (DHE) and mitochondrial (Mito-SOX) ROS; scale bar = 200 µm.(d and e) Mean ± SEM of fluorescence intensity corresponding to (c).(f and g) NOX2 inhibition abolishes MPP + -induced cell death.(f) Fluorescent images of cells treated as in (a) but stained for cell death (Hoechst and PI); scale bar = 125 µm.(g) Mean ± SEM of % PI positive dead cells corresponding to (f).Mean data in (b, d, e, and g) are from three independent experiments (n = 3); **p ˂ 0.01; ***p ˂ 0.001 from One-way Anova, post-hoc Tukey test.(h) Schematic summary of the findings.

Figure 8 .
Figure 8.A distinct ROS generating signalling circuit (right panel) determines the pathological outcome.Unlike the mechanism responsible for the generation of ROS required for cell homeostasis and adaptation to stress (left panel), this signalling circuit involves activation of TRPM2-NOX2 duo to rise cytosolic Ca 2+ to supra-physiological concentration to generate Zn 2+ signals that, by inhibiting complex III, triggers excessive mitochondrial ROS production, leading to pathological outcome (right panel).
2 DCF-DA or DHE, and mitochondrial ROS was detected using MitoSOX Red (M36008, Invitrogen).Following the desired treatments, media were replaced with HBSS containing 10 µM H 2 DCF-DA or 5 µM DHE or 5 µM MitoSOX Red and incubated for 30 min at 37 °C.Cells were counter-stained with Hoechst 33,342 and washed three times with HBSS, before imaging using the EVOS FL Auto 2 Imaging System fitted with a 20 × or 40 × objective and DAPI (excitation, 357 nm; emission, 470 nm for Hoechst), GFP (excitation, 470 nm; emission 525 nm for DCF) and RFP (525 nm excitation, 593 nm emission for DHE and MitoSOX Red) filters.In some experiments, LSM700 inverted confocal microscope fitted with 63x/1.4NA oil objective (excitation, 500 nm; emission, 582 nm) was used.